It has recently become clear that, in terms of digestion, oligopeptides have better absorption rates and a better balance of amino acids following absorption than do mixtures of free amino acids (Rakuno Kagaku-Shokuhin no Kenkyu Dairy Sciences and Food Research!, Vol. 39, No. A, p. 283 (1990)). It is also clear that there has been a rapid increase in patients suffering from allergies induced by food proteins, and that many allergies caused by whey protein, particularly .beta.-lactoglobulin, have appeared, particularly in infants (Rakuno Kagaku-Shokuhin no Kenkyu Dairy Sciences and Food Research!, Vol. 39, No. A, p. 283 (1990)). There is a need to reduce the antigenicity of whey protein in food products for infants or to essentially remove whey protein antigen from food products for infants.
The hydrolysis of whey protein has been widely adopted as a means for reducing the antigenicity of whey protein in food products for infants or for essentially removing whey protein antigen from food products for infants, but hydrolysates with an extremely low percentage of free amino acids often taste bad, which can cause problems when they are ingested. Hydrolysates of whey protein are also sometimes unstable against heat, resulting in precipitation in a liquid state, browning, and other such disadvantages which have posed problems when conventional hydrolysates are used as oral nutrients and the like.
Preventing oxidation is another major issue when whey protein hydrolysates are used in food products, particularly in fatty foods (such as powder milk prepared for infants, which contains as much as 27% fat per 100 g). That is, the balance between saturated and unsaturated fatty acids is considered from a nutritional standpoint in food products that contain fats, but a drawback is that unsaturatio d fatty acids are readily oxidized. DHA and the like which are contained in large amounts in the biological membranes of brain, neural, and retinal tissue and which have recently been believed to play a role in the manifestation of their functions release an extremely strong oxidation odor once they are oxidized, having a markedly adverse effect on product quality, and there is thus a need to prevent their further oxidation.
Ingested amino acids are degraded by transglutaminase, glutamate dehydrogenase, and the like to produce ammonia, but the ammonia thus produced is toxic and must be immediately treated by the liver, so it is essential that no ingested foods contain ammonia. In this regard, it is extremely important that whey protein hydrolysates contain no ammonia.
In view of the nitrogen equilibrium in mature animals, nitrogen should be ingested in an amount corresponding to the minimum metabolic amount of nitrogen, but nitrogen is ineffective when just given in the form of ammonia, and it must be ingested in the form of essential amino acids. Foods that are ingested must therefore contain the necessary amounts of essential amino acids.
Many methods for producing hydrolysates by the enzymolysis of whey proteins have been developed in view of the nutritional and physicochemical background of the proteins and amino acids described above. Several examples are given below.
1) Whey protein is hydrolyzed either with two enzymes, one being a Bacillus subtilis-derived endopeptidase and one being trypsin, or with three enzymes, including a Bacillus subtilis-derived endopeptidase, trypsin, and chymotrypsin, to obtain an oligopeptide mixture with a molecular weight of no more than 2,000 daltons, antigen persistence of no more than 10.sup.-4, and a free acid content of no more than 5% (Japanese Laid-Open Patent Publication 4-248959). PA1 2) Whey protein is hydrolyzed with an alkali protease to obtain a hydrolysate which has at least 75 mol % dipeptides and tripeptides, which has a free amino acid content of less than 5%, which consists of at least four amino acids, and which has less than 20 mol % peptides with an average chain length of 6.2 (Japanese Laid-Open Patent Publication 5-505304). PA1 3) Whey, casein, and soybeans are hydrolyzed with pepsin, trypsin, and chymotrypsin, and ultrafiltration of the product results in an oligopeptide which has a molecular weight of no more than 60,000 daltons and which is 40 to 60% peptides containing 4 to 10 amino acids (Japanese Laid-Open Patent Publication 3-187348). PA1 4) Whey protein is hydrolyzed by thermal denaturation at a pH of 6 to 10 and a temperature of 60 to 80.degree. C., and the enzyme is inactivated by heat to obtain a hydrolysate which has a molecular weight of no more than 10,000 daltons, main peaks of 1,000 to 5,000, an average peptide chain length of 3 to 8, a free amino acid content of no more than 20%, and no more than 1/10,000 .beta.-lactoglobulin antigenicity (Japanese Laid-Open Patent Publication 4-112753). PA1 5) Whey protein is hydrolyzed with trypsin, .alpha.-chymotrypsin, and Aspergillus and Bacillus enzymes to obtain a low allergenic peptide with a molecular weight of no more than 10,000 daltons and the capacity to induce oral immunotolerance (Japanese Laid-Open Patent Publication 5-5000). PA1 6) Casein is hydrolyzed with an acidic protease and is hydrolyzed with a neutral peptidase to obtain a peptide with a molecular weight of no more than 3,000 daltons, a free amino acid content of 30 to 55%, no more than 1 in 10,000 parts .alpha..sub.s -casein in an ELISA inhibition test for .alpha..sub.s -casein, and a 5% solution bitterness organoleptic value no greater than that corresponding to a 0.04% aqueous solution of caffeine (Japanese Laid-Open Patent Publication 6-113893). PA1 7) A method has been disclosed in which whey is hydrolyzed at a pH of 5 to 11 using a neutral protease (Aspergillus) and then heated at a pH of 2 to 4, and the precipitate is removed to obtain 50% dipeptides and tripeptides (Japanese Patent Publication 5-82412). PA1 a) methods for producing the target peptide mixture by hydrolyzing the starting material protein with only an endopeptidase to minimize the production of free amino acids; PA1 b) methods for hydrolyzing the starting material protein with a combination of an endopeptidase and an exopeptidase to produce a peptide mixture which conversely contains a prescribed amount of free amino acids; PA1 c) methods for purifying and fractionating such peptide mixtures into a target peptide mixture by separation such as ultrafiltration (UV), reverse osmosis (RO), gel filtration, and ion exchange resin methods; and the like. PA1 1) A method that has been disclosed is characterized in that enzymolysis is brought about for 0.5 to 10 hours in an aqueous system containing both an endo-type protease and exo-type protease to obtain oligopeptides with an average chain length of 3 to 10 and with very little bitterness (Japanese Laid-Open Patent Publication 62-143697). PA1 2) In another method that has been disclosed, any starter protein starting material is dispersed to between 5 and 20% (w/v) in water, the pH is adjusted to between 1 and 4 with an acid, and enzymolysis is brought about as two or more acidic proteases are added simultaneously or consecutively to suppress the production of free amino acids for 8 to 72 hours at a temperature of 25 to 60.degree. C., so as to produce a low molecular weight peptide composition (Japanese Patent Publication 57-45560). PA1 3) A method for producing a casein partial hydrolysate by controlling the flow ratio of the casein solution has been disclosed for methods involving the partial hydrolysis of a casein solution using a column packed with an immobilized enzyme (Japanese Patent Publication 3-31421). PA1 4) A method for producing a proteolytic product in which the production of insoluble products is prevented is characterized in that a dissolving promoter and a protein or a substance containing a protein are mixed and dissolved in dissolving water or the like, and one or more proteases is or are added to bring about digestion and thus produce a proteolytic product, during which time the viscosity of the solution obtained following the addition of at least the initial proteolytic product is measured over time, and the digestion reaction is stopped before the viscosity begins to fall following a substantial increase (Japanese Patent Publication 3-58252). PA1 5) A method for inactivating the enzyme by heat treatment when the ratio of hydrolysis reaches 5 to 25% has been disclosed for methods of producing peptide mixtures which are the protease hydrolysates of animal milk .kappa.-casein-derived glycopolypeptide, with a Fisher value ranging from 30 to 60 (Japanese Laid-Open Patent Publication 2-300137). PA1 6) A method for measuring peptide concentration with a peptide sensor and a method for measuring the average chain length of peptides have been disclosed for methods of producing hydrolysates in the hydrolysis of proteins using immobilized proteases (edited by Shokuhin Sangyo Baioriakuta Shisutemu Gijutsu Kenkyu Kumiai Association for Research on Food Industry Bioreactor Systems!, Jissen Baioriakuta Practical Bioreactors!, pp. 166-184, published by Association for Research on Food Industry Bioreactor Systems (1990)). PA1 7) It has been disclosed that gluten hydrolysates with excellent foaming stability can be produced by measuring the hydrophobicity of gluten hydrolysates using reverse phase high performance liquid chromatography (high performance liquid chromatography is henceforth HPLC) in methods for producing gluten hydrolysates by hydrolyzing wheat gluten using immobilized proteases (Jissen Baioriakuta Practical Bioreactors!, pp. 106-126, published by Association for Research on Food Industry Bioreactor Systems (1990)). PA1 8) It is known that the free amino acid content in the target product is measured during processing and management of the fermentation of amino acids such as lysine and glutamic acid and the production of fermented foods such as soy sauce and miso sauce (Shokuhin Kogyo Food Industry!, Vol. 34, No. 16, pp. 1-11 (1991)). PA1 9) A method has been disclosed for producing a low molecular weight peptide with no antigenicity, a molecular weight of no more than 1,000 daltons, a free amino acid content of no more than 20%, and an aromatic amino acid content of no more than 1.0% of the total amino acids by recovering the peptide components through gel filtration following hydrolysis until the starting material protein is no longer found to be antigenic and until the aromatic amino acids contained in the starting material protein are at least 90% free amino acids (Japanese Laid-Open Patent Publication 2-138991). PA1 10) A method that has been disclosed for producing a peptide mixture from cow milk whey protein is characterized in that cow milk whey protein is brought into contact with a protease capable of simulating the digestion of proteins occurring in the body, so as to continue the hydrolysis until the product contains virtually no residual protein, that is, until it contains no nitrogen capable of precipitating in 12% trichloroacetic acid, and until a peptide mixture is obtained in which at least 50% of the peptides contain 2 to 5 amino acids, with a free amino acid content of no more than 15% (Japanese Patent Publication 62-61039). PA1 11) A cosmetic and topical skin agent that has been disclosed is characterized in that the hydrolysate of a protein has a molecular weight of no more than 1,000, with at least 90% of the aromatic amino acids being free amino acids, has action in activating the growth of human skin cells, and has no lactoprotein antigenicity (Japanese Laid-Open Patent Publication 4-26604). PA1 a) less than 1% (by weight) of the total hydrolysate consists of fractions having a molecular weight of between 5,000 and 10,000 daltons; PA1 b) the residual antigenic activity is no more than 10.sup.-5, as determined by the ELISA inhibition test using anti-whey protein sera; PA1 c) the amount of free amino acids is 10 to 15% (by weight) with respect to the total amount of amino acids in the hydrolysate; PA1 d) the amount of free lysine is 12 to 20% (by weight) with respect to the total amount of lysine contained in the whey protein; PA1 e) the ammonia content is no more than 0.2% (by weight); PA1 f) the transmittance of a 10% (by weight) solution is at least 98%, as determined at 540 nm using a 1-cm cell; PA1 g) no precipitation results when a 5% (by weight) solution with pH 4 to 7 is heated for 10 minutes at 120.degree. C.; and PA1 h) it has antioxidant activity. PA1 a) As indicated in FIG. 1, less than 1% (by weight) of the total hydrolysate consists of fractions having a molecular weight of between 5,000 and 10,000 daltons. It contains no fractions with a molecular weight of 10,000 daltons or more. At least 70% of the fractions have a molecular weight of less than 1,000 daltons. It has peaks at a molecular weight of 500 daltons and a molecular weight of 1,000 daltons. The number average molecular weight is 300 to 400 daltons, and the weight average molecular weight is 600 to 800 daltons. PA1 b) As shown in FIG. 2, the residual antigenic activity is no more than 10.sup.-5, and preferably no more than 10.sup.-6, as determined by the ELISA inhibition test using anti-whey protein sera. PA1 c) The amount of free amino acids is 10 to 15% (by weight), and preferably 11 to 13% (by weight), with respect to the total amount of amino acids in the hydrolysate. PA1 d) The amount of free lysine is 12 to 20% (by weight), and preferably 14 to 17% (by weight), with respect to the total amount of lysine contained in the whey protein. PA1 e) The ammonia content is no more than 0.2% (by weight), and preferably no more than 0.1% (by weight). PA1 f) The transmittance of a 10% (by weight) solution is at least 98%, as determined at 540 nm using a 1-cm cell. PA1 g) No precipitation results when a 5% (by weight) solution with pH 4 to 7 is heated for 10 minutes at 120.degree. C. PA1 h) As shown in FIG. 3, it has antioxidant activity equal to or greater than that of .alpha.-tocopherol, a well-known antioxidant. FIG. 3 indicates the antioxidant activity of the whey protein hydrolysate of the present invention obtained in Example 1. The vertical and horizontal axes indicate the residual antioxidant capacity and the time, respectively. In the figure, the diamonds, plus signs, and boxes indicate the whey protein hydrolysate of the present invention, .alpha.-tocopherol, and a control (sample or preparation added), respectively.
Peptide mixtures obtained by the proteolysis of animal proteins (animal milk, eggs, meat, fish, and the like) or vegetable proteins (soybeans, wheat, and the like) are known to have properties such as thickening, foaming, antioxidant, digestive, mineral solubilizing, and low antigenicity properties, as well as epithelial cell growth factor, cell growth factor, calcium absorption promoting function, opioid-like activity, and other such physiologically active functions (Shokuhin to Kaihatsu Food Products and Development!, Vol. 26, No. 11, pp. 28-36 (1991)). They are an indispensable material in the manufacture of meat, fish paste, breads, sweets, mineral fortified food products, infant food products, sports beverages, general health foods, enteric nutrients, food products to combat protein allergies, special nutritional food products, medical drugs, and the like.
Methods for producing the peptide mixtures used in the manufacture of these food products and medical drugs vary, depending on the application, and can be broadly divided into:
Phenylketonuria (henceforth PKU) is a congenital metabolic disorder in which phenylalanine (henceforth Phe) accumulates in the blood, resulting in neurological disorders and developmental disorders, due to a congenital deficiency of phenylalanine hydroxylase which converts the amino acid phenylalanine into tyrosine. Patients suffering from PKU must accordingly strictly limit the amounts of Phe ingested under the supervision of a physician so as to avoid the accumulation of Phe in the body.
Since, on the other hand, Phe is a common amino acid that is usually contained in an amount of about 3 to 5% in proteins, patients of PKU have had to ingest part or all of the protein component of food products or infant milk preparations by substituting them with amino acid mixtures containing no Phe. These types of amino acid mixtures, however, suffer from drawbacks such as the disagreeable taste characteristic of amino acids and the diarrhea which results from high osmotic pressure in the intestines. There is thus a desire on the part of patients, their families, and physicians for a palatable source of protein serving as a suitable food therapy for patients of PKU.
A method involving the use of .kappa.-casein glycomacropeptide (henceforth GMP) as a protein source for patients of PKU has been disclosed as one such method (Japanese Laid-Open Patent Publication 4-126051). The amino acid sequence of GMP contains no Phe, the molecular weight is a substantial 8,000 daltons, and the problem of elevated osmotic pressure is virtually absent, making this substance an effective source of protein for patients of PKU. However, the isolation of GMP is extremely complicated and is unsuitable for industrial production. Moreover, recent nutritional findings have made it clear that oligopeptides are more readily digested than proteins.
Another example of the use of oligopeptides as a source of protein for patients of PKU is the method in which proteolysis is brought about with a protease, fractions containing no Phe are recovered by gel filtration, and the resulting low phenylalanine peptide (henceforth LPP) is used (Journal of Food Science, Vol. 41, pp. 1029-1032 (1976), and Japanese Laid-Open Patent Publication 61-68426).
In another method that has been disclosed, proteins are treated with an exopeptidase or are treated with an exopeptidase following treatment with an endopeptidase, activated carbon is used to adsorb polypeptides containing virtually no aromatic amino acids as well as low molecular weight peptides having terminal free aromatic amino acids or aromatic amino acids, and the low molecular weight substances are separated using reverse osmosis membranes or ion exchange electrodialysis membranes to produce LPP (Japanese Patent Publication 2-54070). This method has made the industrial manufacture of LPP possible.
The following conventional techniques are examples for producing peptides by the hydrolysis of starting material proteins using protease.
Although decreases in the antigenicity of the whey protein hydrolysate, improvements in the taste, the free amino acid content, the molecular weight distribution, or the like are considered in the aforementioned conventional techniques, no consideration has been given to the ammonia content and the antioxidant action of the whey protein hydrolysate. A drawback in the past has thus been that whey protein hydrolysates could not be used for a wide range of food products.
Moreover, as indicated in the aforementioned conventional techniques, when peptide mixtures are produced by hydrolyzing starting material proteins with proteases, the end point of the hydrolysis reaction has been determined by measuring the hydrolysate viscosity, ratio of hydrolysis, degree of hydrophobicity, or the like using the reaction time, the protein solution flow ratio , and the like as indices, but it is extremely difficult in these methods to achieve an accuratio grasp of the changing physicochemical state of the hydrolysate, particularly the free amino acid content, and a fatal drawback which needs to be remedied in conventional methods for producing peptide mixtures is that each batch which is manufactured results in a peptide mixture with a different free amino acid content and composition, leading to peptide mixtures of inconsistent quality.
Furthermore, to bring about enzyme reaction with good reproducibility, it is necessary to strictly control the reaction temperature, pH, enzyme titer, substratio concentration, and the like, which is impractical for operations on an industrial scale and makes it extremely difficult for practical purposes to obtain a consistent free amino acid ratio.
This means that the production of high quality LPP is also plagued by the following fatal drawbacks. As described above, in LPP manufacturing methods which include a step involving the hydrolysis of proteins using proteases, the method for lowering the Phe content of the peptide mixture is based on the principle that a sufficient amount of Phe is freed from the proteins by the hydrolysis of the proteins using the protease, and that the freed Phe is removed by gel filtration, activated carbon adsorption, or the like, which means the control of the protease-based hydrolysis of the protein is an important technique for producing high quality LPP.
That is, when Phe is insufficiently freed, a low amount of Phe is removed by gel filtration or activated carbon treatment following the enzymolysis, resulting in a peptide mixture with a higher Phe content, which is unsuitable for use by patients suffering from PKU. When too much Phe is freed (in other words, when enzymolysis has progressed too far), there is a higher ratio of free amino acids other than Phe, resulting in extremely poor taste, and other drawbacks include extremely poor therapeutic effects because of the unpleasant taste, the incidence of diarrhea, and the like, so these problems currently need to be resolved. That is, an extremely important issue for producing high quality LPP is to consistently ensure a certain content of free Phe in peptide mixtures obtained by the hydrolysis of proteins using proteases.